This application claims priority to International Application No PCT/CA99/00673, filed Jul. 23, 1999 (Publ. No. WO 00/07618), and Canadian Application No 2,239,248, filed Jul. 31, 1998.
The mechanisms for establishment of latent herpes simplex virus infection in neurons and the subsequent reactivation are very poorly understood. Recent studies have shown that the pattern of gene expression during reactivation is not similar to the one seen in the lytic cycle: the expression of early (E) genes, notably the gene for the subunit 1 (R1) of ribonucleotide reductase, begins several hours before detectable expression of the immediate early (IE) genes (1-3). HSV can be reactivated by numerous stress conditions including NGF deprivation, hyperthermia and cadmium (4) which are also known to induce neuronal cell apoptosis. Therefore, it might be advantageous for the virus to encode protein(s), which are able to block the apoptotic pathways activated by these stimuli. In addition, such proteins could be important to counteract the action of cytotoxic T lymphocytes (CTL) which prevent virus dissemination in cells of the mucosal epithelia where it replicates after being released from neurons (5).
The HSV ribonucleotide reductase converts ribonucleoside diphosphates to the corresponding deoxyribonucleotides and plays a key role in the synthesis of viral DNA (reviewed in (6). The association of two subunits, R1 and R2, the former of which contains the active site, forms the holoenzyme. The R1 subunits of HSV-1 and HSV-2 possess an NH2 domain of about 350 amino acids. This is a unique feature which is not found in R1 of other species, including those of other herpes viruses (7, 8). The role of HSV ribonucleotide reductase has been extensively studied with ribonucleotide reductase null mutants. Studies first done with cultured cells showed that the enzyme is required for efficient replication in non dividing cells. Subsequently, works using animal models demonstrated that the enzyme is required for efficient pathogenicity, is essential for viral reactivation from the neurons, but is not essential for the establishment of latency (9-16). The observations that a mutant virus bearing a deletion of the reductase domain of the R1 gene (hrR3) exhibited the same phenotype in cell culture or in animal models as a virus with a deletion of both the NH2 and the reductase domains (ICP6xcex94) has suggested that the NH2 domain may play only a minor role in viral pathogenesis (9, 10, 13). However, as viral mutants which contain deletions only of the R1 NH2 domain have not yet been characterized for their capacity to reactivate, an important role of this domain in HSV reactivation could have been masked by the ribonucleotide reductase deficiency of the two mutants which by itself completely prevents viral replication in the latently infected neurons.
The view that a protein kinase activity or at least an autophosphorylation activity could be intrinsic to the unique NH2 domain of the R1 had been supported over the past ten years by several studies (17-24). However, it has been recently challenged by extensive works showing that R1 does not possess an autophosphorylation activity but rather is a good substrate for copurifying protein kinases (25, 26)]. Our unsuccessful attempts to select standard recombinant Ad for an HSV-2 bearing a complete deletion of its NH2 domain, xcex94R1, have led us to suspect that this protein could be cytotoxic and thus, to develop a transfer vector (pAdTR5) that utilizes a tetracycline-regulated expression cassette. Hence, a recombinant Ad with tetracycline-regulated expression of xcex94R1, Ad5TR5-R1 (xcex942-357), was readily obtained and the pro-apoptotic potential of the xcex94R1 protein was demonstrated by infecting cells expressing the tet-regulated transactivator (tTA) (27). This is therefore a strong suggestion that the N-terminal fragment of the R1 protein is an anti-apoptotic protein per se. A broad anti-apoptotic activity would make the N-terminal R1 protein a candidate of choice as an anti-apoptotic agent, alone or in combination with other anti-apoptotic agents.
The anti-apoptotic activity of the RI protein N-terminal domain is exploitable per se, as well as in the making of a virus or of a viral vector that would be less virulent. The xe2x80x9ccassettexe2x80x9d RI N-terminal would therefore be inserted in such a virus or viral vector. On the opposite a herpes virus encoding the RI without the anti-apoptotic domain could be used to destroy undesirable cells like cancer cells. In both cases, gene therapy is envisaged to deliver one or the other RI proteins as well as to co-deliver other proteins of interest.
The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which is expressed very early after viral reactivation, possesses an N-terminal domain of about 350 amino acids of unknown function. Using an adenovirus (Ad) inducible system we had demonstrated that a complete deletion of this domain produces a cytotoxic protein. We now report that apoptotic death induced by this trncated R1 could be completely prevented by coexpression of the full-length R1. The R1 anti-apoptotic activity was further substantiated by showing that expression of this protein at low level can completely block apoptosis induced either by TNF-receptor family triggering in the presence of cycloheximide (CHX) or by Fas-L coexpression with an Ad recombinant. In both cases, the protection was lost when inhibiting tTA function with the tetrycline analog doxycycline shut down R1 expression. A level of R1 of 0.005% total cell protein was sufficient for half-maximal protection against TNFxcex1+CHX. By monitoring caspase 8 activation either by immunoblot with an antiserum visualizing the inactive 56-kDa proform and the active 18-kDa species or by an in vitro assay using ETD-AFC as caspase 8 specific fluorescent substrate, we found that the strong activation of caspase 8 induced either by TNF-xcex1+CHX or Fas-L expression was prevented by the R1 protein. Finally, using an HSV-1 R1 deletion mutant, ICP6xcex94, we obtained direct evidence for the importance of HSV-R1 in protecting HSV-infected cells against cytokine-induced apoptosis. These results show that, in addition to its reductase function which is essential for viral reactivation, the HSV R1 could contribute to viral propagation by preventing apoptosis induced by the immune system. The N-terminal domain by itself is as anti-apoptotic as the whole R1 protein. An anti-apoptotic agent and a composition derived therefrom are described and claimed.
The present invention relates to the new use of the subunit 1 of HSV ribonucleotide (RI) reductase, a variant or a part thereof having a functional anti-apoptotic N-terminal domain, as an anti-apoptotic agent.
The present invention further relates to an anti-apoptotic agent comprising the N-terminal 357 amino acids of the RI sub-unit, a variant thereof or anti-apoptotic part thereof. It is another object of this invention to provide an anti-apoptotic composition comprising RI, the N-terminal portion of RI, a variant or a part thereof having an anti-apoptotic activity. A variant is defined as a molecule having been subjected to mutation by substitution, deletion or addition, which mutation has no deleterious effect on the anti-apoptotic activities. The sequences of the RI subunit of HSV-1 and -2 are disclosed in (7-8). Similarity or identity of more than 50%, preferably of more than 70%, even more preferably of more than 90%, would achieve a functional variant provided that the mutation is not directed to an amino acid residue essential for the activity. Due to codon degenerescence, a lesser degree of conservation is needed for the nucleic acids used for the purpose of providing functional variants.
Such a composition may also comprise another anti-apoptotic agent. Such agent includes but is not limited to anti-caspase molecules (such as enzymatic inhibitors, inhibitors of synthesis or activation, antibodies or other ligands), anti-cytokines such as anti-TNFxcex1 molecules (antagonists, degradation activators or inhibitors of synthesis or of secretion, antibodies or other ligands), a bcl-2 protein, homologue or mimetic, anti-Fas-L molecules (antagonists, degradation activators, or inhibitors of synthesis or of secretion), and agents interfering with the recruitment, binding and/or activity of cytotoxic white blood cells at a diseased tissue site.
We demonstrated, by coinfection experiments with full-length R1 and xcex94R1 recombinants to obtain coexpression of both proteins, that the NH2 domain of the HSV-R1 has an antiapoptotic function able to protect the cells against death triggered by the expression of the reductase domain alone. More importantly, it was found that the full-length R1 has a broad anti-apoptotic potential as it is able to protect cells against not only xcex94R1 but also against death induced by TNF-xcex1 and Fas receptor triggering. Caspase 8 activation, which occurs with both types of death-inducing signal, was impaired by R1 expression. Finally, using an HSV-1 R1 deletion mutant, ICP6A, we obtained direct evidence for the importance of HSV-R1 in protecting HSV-infected cells against cytokine-induced apoptosis.